Construction of a Novel Bifunctional by Two Point Mutations of the H2-

John DelValle,* Ira Gantz,t Lidong Wang,* Yi-Jun Guo,* Gerd Munzert,* Takao Tashiro,* Yoshitaka Konda,* and Tadataka Yamada** Departments of *Internal Medicine, tSurgery, and tPhysiology, University of Michigan Medical Center, Ann Arbor, Michigan, U.S.A.

ABSTRACT Background: H2-histamine receptors mediate a wide expression vector, CMVneo, and stably transfected into range of physiological functions extending from stimu- Hepa cells and L cells. The biological activity of histamine lation of gastric acid secretion to induction of human and epinephrine on the expressed receptor was exam- promyelocyte differentiation. We have previously cloned ined by measurement of cellular cAMP production and the H2-histamine receptor gene and noted that only inositol trisphosphate formation. three amino acids on the receptor were sufficient to Results: Hepa cells transfected with the Ala'86-Serl87 define its specificity and selectivity. Despite only modest mutant H2 receptor demonstrated a biphasic rise in overall amino acid homology (34% amino acid identity cAMP in response to epinephrine with an early phase and 57.5% similarity) between the H2-histamine recep- (ED50 10- " M) that could be inhibited by both pro- tor and the receptor for another monoamine, the ,B2- pranolol and cimetidine. Epinephrine also induced IP3 , there is remarkable similarity at generation in the same cells, a biological response that is their critical ligand binding sites. We hypothesized that, if characteristic of activation of the wild-type H2 but not of the specificity and selectivity of both receptors are in- the ,B-adrenergic receptor. L cells transfected with the vested in just three amino acids, it should be possible to Ala'86-Serl87 mutant H2 receptor also responded to epi- convert one of the receptors into one that recognizes the nephrine in a cimetidine and propranolol inhibitable ligand of the other by simple mutations at only one or manner. two sites. Conclusions: We converted the H2-histamine receptor Material and Methods: We explored the effect of two into a bifunctional one that has characteristics of both single mutations in the fifth transmembrane domain of histamine and adrenergic receptors by two simple mu- the H2-histamine receptor, which encompasses the sites tations. These results support the hypothesis that ligand that determine H2 selectivity. The canine H2 receptor specificity is determined by only a few key points on a gene was mutated at Asp'86 and Gly'87 (Asp'86 to Ala'86 receptor regardless of the structure of the remainder of and Gly'87 to Ser'87) by oligonuceotide directed mu- the molecule. Our studies have important implications tagenesis. The coding region of both the wild-type and on the design of pharmacological agents targeted for mutated H2 receptors was subcloned into the eukaryotic action at physiological receptors.

INTRODUCTION cloned the H2 -histamine receptor gene (1) and The availability of genes encoding an increas- noted that only three amino acids on the recep- ingly wide array of receptors for regulatory sub- tor were sufficient to define the specificity and stances has led to insight into the molecular basis selectivity of the receptor (2). By extrapolation of of ligand-receptor interactions. Recently, we these findings, it is conceivable that ligand spec- ificity is determined by only a few key points on Address correspondence and reprint requests to: John a receptor regardless of the structure of the re- DelValle, 6520 MSRBI, Ann Arbor, MI 48109-0682. mainder of the molecule. We have previously

280 Copyright @ 1995, Molecular Medicine, 1076-1551/95/$10.50/0 Molecular Medicine, Volume 1, Number 3, March 1995 280-286 J. DelValle et al.: Construction of a Bifunctional H2-Histamine Receptor 281

catechol ring (4). The H2-histamine receptor has acid and threonine residues in a 0 aspartic compa- III Psp VI 9\ 'IC -0o rable but not identical region of its fifth trans- CH2 membrane domain (Asp'86-Thr'90) that are im- portant for binding to the imidazole ring of 1--1 i ..H- 0f histamine (2). Thus, there is remarkable similar- ,0-~H-N o-H- O=C X_H ity in the ligand binding sites for histamine and ,CH2 \ Gly Va catecholamines on the H2-histamine and ,32-ad- A~6 V renergic receptors, respectively, despite the rela- FIG. 1. Schematic drawing of the interaction tive lack of in the rest of their of histamine with the H2-histamine receptor homology struc- ture. In light of this observation, we explored the The amino acid sequences of the homologous re- gions of the H2-histamine and f2-adrenergic recep- effect of two simple mutations (Asp'86 to Ala'86 tors (amino acids 186-190 and 203-207 on the re- and Gly'87 to Serl87) on the pharmacological spective receptors) are indicated below. The two characteristic of the H2-histamine receptor. We alterations made in the mutant receptor are indi- reasoned that these amino acid substitutions, de- cated with italics. (From Ref. 2.) signed to make the putative ligand binding site of the H2-histamine receptor resemble more closely that of the 02-adrenergic receptor, would con- vert the H2-histamine receptor into one that rec- noted a remarkable similarity between the H2- ognizes catecholamines. histamine receptor and the receptor for another monamine, the P2-adrenergic receptor, at their critical ligand binding sites. However, there is otherwise only modest overall amino acid ho- METHODS mology (34% amino identity and 57.5% similar- Mutagenesis and Expression ity), and, furthermore, there is no evidence to indicate cross-reactivity of histamine at ,B2-ad- For our studies, the canine H2-histamine recep- renergic receptors or, conversely, catechol- tor gene was subcloned into the sequencing vec- amines at H2-histamine receptors. We reasoned tor Ml 3 and used as a template for the synthesis that, if our hypothesis that the specificity and of DNA with specific mutations according to the selectivity of both receptors are invested in just method of Kunkel (5). A polymerase chain reac- three amino acids is correct, it should be possible tion (PCR) strategy was used to subclone the to convert one of the receptors into one that coding region of both the wild-type and mutated recognizes the ligand of the other by simple mu- H2-histamine receptors into the eukaryotic ex- tations at only one or two sites. In the present pression vector CMVneo, as previously described studies, we provide support for this hypothesis (2,6). Hepa cells (derived from a rat hepatoma) by demonstrating that two amino acid substitu- and L cells (derived from a mouse fibroblast-like tions are sufficient to convert the H2-histamine cell) were then transfected with the receptor/ receptor into a novel bifunctional receptor that CMVneo constructs by calcium phosphate copre- has characteristics of both histamine and adren- cipitation (7) and clones were selected in media ergic receptors. containing the neomycin analogue G-418. The For these studies, we took advantage of the expression of receptor RNA was examined by fact that key areas of homology between the Northern blot analysis and clones expressing H2-histamine and f32-adrenergic receptors sug- roughly equivalent amounts of receptor RNA gest specific amino acids in the third and fifth were used for further studies. transmembrane domains of the receptors that might be important for ligand recognition and binding (Fig. 1). Both receptors contain an aspar- cAMP Assays tic acid in the third transmembrane domain that Intracellular cyclic 3', 5'-adenosine monophos- serves as a counter anion to the cationic amine phate (cAMP) accumulation was measured using moiety of histamine and catecholamines (2,3). In a cAMP Assay Kit (TRK 432; Amersham, Arling- the fifth transmembrane domain, 32-adrenergic ton Heights, IL, U.S.A.). Cells transfected with receptors contain two serine moieties (ser204 and the receptor genes were grown to confluence in Ser207) that are thought to serve as points for 12-well (2.4 X 1.7 cm) tissue culture plates. The hydrogen bonding to the hydroxyl groups in the cells were maintained in Dulbecco's modified 282 Molecular Medicine, Volume 1, Number 3, March 1995

Eagle's medium (DMEM; Gibco, Grand Island, (a) (b) (c) Noafra,sfected Wid Type Jal36Sert87 Mutant NY) containing 4.5 g/100 ml glucose, 10% fetal H2 Receptor H2 Receptor calf serum, 100 units/ml penicillin and strepto- mycin, 1 mM sodium pyruvate and 1 mg/ml of HEPA cells geneticin. For assays, this media was removed and cells were washed twice with Earle's bal- anced salt solution (EBSS) containing 10 mM HEPES (pH 7.4), 1 mM glutamine, 26.5 mM - 11 10 9 8 7 6 5 4 11 16 9 8 7 6 5 4 -1110 9 8 7 6 5 4 sodium bicarbonate, and 100 mg/ml bovine se- [Epinephrine] (-log M) rum albumin. An aliquot (0.5 ml) of EBSS was FIG. 2. Effect of epinephrine on cAMP genera- placed into each well along with 5 ,ul of 2 X 10-2 tion in transfected Hepa cells M isobutylmethylxanthine. Varying concentra- (a) Epinephrine dose-dependently stimulated cAMP tions of agonist were added and the cells were generation in nontransfected Hepa cells (EC50 = incubated for 30 min at 37°C. Ice-cold 30% tri- 5 X 10-8 M, maximum response = 278 ± 44% chloracetic acid (500 ,ul/well) was added to above control, means ± SEM, n = 4). This effect stop was inhibited by propranolol (10-5 M) but not by the reaction and precipitate cellular protein. The cimetidine (10-5 M). In Hepa cells transfected with cells were scraped and transferred to 16 X 150 the wild-type H2 receptor (b), epinephrine stimu- mm glass tubes then placed on ice for 30 min. lated cAMP generation in a biphasic fashion. The The precipitate was then centrifuged for 10 min stimulatory effect of low-dose epinephrine (10 "to at 1,900 x g and the supernatant was ether 10-9 M) on cAMP formation was inhibited by cime- tidine and not by propranolol, while the effect ob- extracted, lyophilized, and resuspended in 50 served with higher concentrations of epinephrine mM tris, 2 mM EDTA (pH 7.5). cAMP content (10-8 to 10-4 M) was inhibited by propranolol and was then measured by competitive binding assay not by cimetidine. (c) The stimulatory action of epi- according to the assay instructions. nephrine on cAMP generation was markedly en- hanced in Hepa cells transfected with the Ala'86- Serl87 mutant H2-histamine receptor (maximum Inositol Phospholipid Assays response = 280 ± 15% above control). The re- sponse to low dose epinephrine (10-" to 10 -9 M) Transfected Hepa cells were grown to confluence was inhibited with both cimetidine and propranolol in 2.4 X 1.7 cm multiwell plates and prelabeled while the response to high epinephrine concentra- with myo inositol at for 2 tions (10 8 to 10 4 M) remained sensitive only to [2-3H] 37°C hr, adding propranolol. Consistent with our previous studies, LiCl (10 ,uM) 10 min before completing the pre- histamine stimulated cAMP generation in Hepa cells incubation period. Histamine or epinephrine expressing both wild-type and mutant H2 histamine were added for varying time intervals and the receptors (data not shown). water soluble cellular products were extracted and separated by ion exchange chromatography on Dowex- 1 resin columns as previously de- scribed using 100 mM increments of ammonium Hepa cells. These stimulatory effects, obtained at formate (8). Inositol monophosphate was eluted relatively high concentrations of epinephrine with 10 mM formic acid/100 mM ammonium (ED50 = 10-7 M), could be inhibited by pro- formate/5 mM disodium tetraborate, inositol pranolol but not cimetidine. Thus, there appears bisphosphate was eluted with 20 mM formic ac- to be a classical ,B-adrenergic receptor on Hepa id/200 mM ammonium formate/5 mM disodium cells. After transfection with the wild type H2- tetraborate, and inositol trisphosphate (IP3) was histamine receptor gene, the Hepa cells demon- eluted with 30 mM formic acid/300 mM ammo- strated a biphasic response to epinephrine. The nium formate/5 mM disodium tetraborate. The later phase of the response presumably repre- fraction containing IP3 consists of a mixture con- sented the endogenous J3-adrenergic receptors taining the 1,4,5 and the 1,3,4 isomers. and mirrored the effect observed in nontrans- fected Hepa cells. In contrast, the initial phase of the cAMP response to catecholamine had a much lower ED50 (<10 "M) and was inhibited with RESULTS cimetidine but not propranolol. These data indi- As shown in Fig. 2, epinephrine, the catechol- cate that catecholamines are capable of having a amine used in these studies, dose-dependently minimal action at the standard H2 receptor, per- stimulated the production of cyclic 3',5'-adeno- haps on the basis of the existing similarity in the sine monophosphate (cAMP) in nontransfected structure of the binding sites of H2-histamine J. DelValle et al.: Construction of a Bifunctional H2-Histamine Receptor 283

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-0 9 8 7 6 5 4 00 8 7 6 5 Histamine (-log M) [Antagonist] (-1ogM) FIG. 3. Effect of histamine on cAMP genera- FIG. 4. Effect of propranolol, cimetidine, di- tion in transfected Hepa cells phenhydramine and thioperamide on epineph- Although histamine had no effect on nontransfected rine stimulated cAMP generation in Hepa cells Hepa cells (data not shown), it stimulated cAMP for- transfected with the mutant Ala'86-Ser187 H2- mation in cells transfected with the Ala'86-Serl87 histamine receptor mutant H2-histamine receptor (ED50 = 3 X 10-8 Propranolol, cimetidine, and diphenhydramine dose- M) in a fashion that could be inhibited by cimetidine dependently inhibited cAMP generation stimulated (10- 5M) and propranolol (10-5 M). Data are repre- by epinephrine with IC50 values of sented as means ± SEM, n = 4. 1 ± 0.3 X 10-7 M, 5 ± 0.45 X 10-7 M, and 5 ± 1.2 X 10-8 M, respectively. Thioperamide did not alter epinephrine stimulated cAMP formation in transfected cells. Data are represented as means + and ,B-adrenergic receptors. Upon transfection SEM, n = 4. with the Ala186-Ser187 mutant H2-histamine re- ceptor, the Hepa cells demonstrated, once again, a biphasic response. However, in this instance, the initial phase was greatly exaggerated and, in the endogenous Hepa cell 3-adrenergic receptor, contrast to the effect obtained with the wild-type could be inhibited with propranolol, cimetidine, H2-histamine receptor, inhibited with either ci- and diphenhydramine, an HI-histamine recep- metidine or propranolol. As depicted in Fig. 3, tor antagonist. Similarly, when the stimulatory histamine dose-dependently stimulated cAMP ligand used was histamine, the same inhibitory formation in Hepa cells transfected with the pattern was observed with the H2-histamine and Ala'86-Ser187 mutant H2-histamine receptor with 13-adrenergic receptor antagonists. The H3-hista- an ED50 (3 X 10 8 M) which is similar to that mine receptor antagonist thioperamide did not obtained in Hepa cells transfected with the wild- inhibit either epinephrine or histamine stimu- type receptor (data not shown). It is of note that lated cAMP formation. Thus, the mutant recep- the stimulatory effect of histamine on Hepa cells tor responded to both adrenergic and histamin- transfected with the mutant H2 receptor was in- ergic stimuli and was inhibited by selective hibited with both cimetidine and propranolol, antagonists for either class of ligands. It is of whereas the effect of this secretagogue on the wild- considerable interest that the Ala'86-Ser187 mu- type H2-receptor was inhibited only by cimetidine tant confers Hi-histamine receptor properties to (data not shown). These data demonstrate that the the H2 receptor. This effect was predicted by our mutant H2-histamine receptor had the bifunc- previous studies demonstrating that Asp'86 of tional properties of H2-histamine and f3-adrenergic the H2-histamine receptor defines H2 selectivity receptors. but is not an essential element in histamine We conducted additional studies to charac- binding (2). terize the bifunctional nature of the Ala'86- One feature that differentiates H2-histamine Ser187 mutant H2-histamine receptor using se- from 13-adrenergic receptors is that while both lective agonists and antagonists. As shown in receptors are capable of generating a cellular Fig. 4, the cAMP response to epinephrine at a cAMP response, only the former has been shown dose of I0- M, which is below the threshold of simultaneously to induce significant turnover of 284 Molecular Medicine, Volume 1, Number 3, March 1995

4uAd% (a) (b) (c) No-Tranosfected WDdTpe Alal8SerIB7 Mutant H2 Receptor H2 Receptor

30 L-ceUs

20 20 Er t - 5 oo 7 9 - 10 9 8 7 6 4 9 8 6 5 4 8 7 6 5 4 [Epinephrine] (-log M) FIG. 6. Effect of epinephrine on transfected L -0 cells Epinephrine produced no increase in cAMP levels in nontransfected L cells (a) and only a small increase -10 at high concentrations in L cells transfected with the 0 20 40 60 wild-type H2-histamine receptor (b). The latter effect Time (sec.) was abolished with cimetidine (10-5 M) but not with propranolol (10 5 M). However, epinephrine FIG. 5. Effect of epinephrine on inositol induced a dose-dependent rise in cAMP generation trisphosphate (IP3) generation in Hepa cells in cells transfected with the mutant Ala186-Ser'87 transfected with the wild-type and Ala'86- H2-histamine receptor (c). This stimulatory effect Ser'87 mutant H2-histamine receptor was inhibited by both cimetidine and propranolol. Epinephrine at a maximal concentration of 10-5 M Histamine increased cAMP levels in L cells trans- induced a time-dependent increase in IP3 generation fected with the wild-type H2-histamine and the in Hepa cells expressing the mutant Ala'86-Serl87 Ala186-Ser'87 mutant receptors, but did not alter H2-histamine receptor but had no such effect on cAMP levels in nontransfected cells (data not nontransfected Hepa cells. Data are represented as shown). Data are represented as means ± SEM, means ± SEM, n = 4. n = 4.

that they do not express endogenous adrenergic membrane inositol phospholipids and mobilize receptors. In very high concentrations, epineph- intracellular calcium (9). Accordingly, to verify rine demonstrated some ability to induce cAMP that the catecholamine response that we were generation via action at the wild-type H2 recep- observing in the Hepa cells transfected with the tor, although this response was inhibited only Ala'86-Ser187 mutant H2-histamine receptor was with cimetidine and not with propranolol. How- a result of action at the mutant histamine recep- ever, a much more characteristic cAMP response tor and not the endogenous catecholamine re- to epinephrine was noted in L cells transfected ceptor, we examined the effect of epinephrine on with the Ala'86-Ser187 mutant H2 receptor, and membrane inositol phospholipid turnover as this response was inhibited completely with both measured by the production of IP3. As shown in propranolol and cimetidine. Histamine also in- Fig. 5, epinephrine (10-5 M) induced IP3 gener- duced a dose-dependent rise in cAMP formation ation in Hepa cells transfected with the Ala'86- in L cells transfected with the Ala186-Ser187 Ser187 mutant H2-histamine receptor. Only a mutant H2 receptor (Fig. 7) and, as with epi- minimal IP3 response was noted at the 10-sec nephrine, this effect was inhibited with both time point in nontransfected Hepa cells. These propranolol and cimetidine. The maximal stim- data confirm that, despite the presence of an ulatory effect of histamine was less than that endogenous ,B-adrenergic receptor, there were seen with the wild-type H2 receptor, but the selective responses of transfected Hepa cells to ED50 was similar in both instances. Nontrans- epinephrine that could be mediated only via ac- fected L cells demonstrated no response to his- tion at a bifunctional histamine receptor. tamine. These findings confirm the bifunc- For further confirmation of the bifunctional tional nature of the receptor that transduces nature of the Ala'86-Ser187 mutant H2-histamine the responses activated by epinephrine and receptor, we transfected both the wild-type and histamine. The relatively broad configuration mutant H2-histamine receptors into a second cell of the dose response curves shown in Figs. 6 line, the L cell. As depicted in Fig. 6, unlike the and 7 may result from the actions of epineph- Hepa cells, nontransfected L cells demonstrated rine and histamine at a bifunctional receptor as no cAMP response to epinephrine, indicating opposed to a single receptor subtype. J. DelValle et al.: Construction of a Bifunctional H2-Histamine Receptor 285

-- Histamine (His) receptors. For example, the alteration of a single 150 -*-- His + Propranolol (10-5M) amino acid has been shown to account for the -0-- His + Cimetidine (10-5M) pharmacological differences between the rat and human 5-hydroxytryptamine lB receptors (10,11) and the canine and human cholecysto- kinin-B/gastrin receptors (12). Similarly, a point C'U 100 mutation in the a2-adrenergic or 5-hydroxy- tryptamine 1A receptors changes their affinity for 13-adrenergic receptor antagonists (13,14). In other studies the receptors for the tachykinins substance P (NK1 receptor) and neurokinin B

50 - (NK3 receptor) have been shown to exhibit al- terations in receptor binding affinity to nonpep- tide antagonists as a result of changes in only one or two of its amino acid residues (15-17). Our experiments provide the first demon- 0 stration that such minor amino acid changes can co 8 7 6 5 4 result in changes in ligand specificity so major Histamine (-log M) that they can cross the gap between entire fam- FIG. 7. Effect of histamine on cAMP genera- ilies of receptors that have only modest structural tion in transfected L cells homology. These findings have important impli- Histamine had no effect on nontransfected L cells, cations in the design of pharmacological agents but stimulated cAMP formation in L cells transfected that are directed at physiological receptors. It is with the wild-type H2 receptor in a fashion that possible, for example, that so-called specific re- could be inhibited by cimetidine (data not shown). ceptor antagonists or agonists may have cross- Histamine also stimulated cAMP formation in L cells over effects on other groups of receptors that transfected with the Ala'86-Ser'87 mutant H2 recep- tor in a dose-dependent fashion (ED50 = 5 x 10-8 would not be predicted on the basis of overall M). The response to histamine was inhibited by both structural homology. Moreover, it may be possi- propranolol and cimetidine. Data are represented as ble to utilize unique agonists or antagonists for means ± SEM, n = 4. targeting ligand delivery to artificially con- structed mutant receptors that are transfected into various tissues in the body. DISCUSSION We have demonstrated that two amino acid sub- stitutions are sufficient to convert the H2-hista- ACKNOWLEDGMENTS mine receptor into a bifunctional one that has characteristics of both the histamine and adren- This work was supported by National Institutes ergic receptors. Our studies suggest that the li- of Health (NIH) Grants RO1DK34306 and gand specific binding properties of a receptor are ROIDK47434, and funds from the University of determined by a surprisingly limited number of Michigan Gastrointestinal Peptide Research Cen- key amino acids in its molecular structure. Al- ter (NIH Grant P30DK34933). Dr. Gantz is a though there is broad structural similarity among recipient of a Veterans Administration Research seven transmembrane G protein-linked recep- Associate Award. We thank Drs. J. Dixon and M. tors, there is relatively low amino acid homology Uhler for their helpful comments on this between families of receptors for different classes manuscript. of agonists. Despite the relatively modest overall structural homology between the H2-histamine and (32-adrenergic receptors, two simple amino acid substitutions are sufficient to convert the REFERENCES former receptor into one that has the ligand rec- 1. Gantz I, Schaffer M, DelValle J, et al. (1991) ognition characteristics of both. Seemingly minor Cloning of the H2 histamine receptor. Proc. amino acid changes have been reported previ- Natl. Acad. Sci. U.S.A. 88: 429-433. ously to have profound effects on the pharma- 2. Gantz I, DelValle J, Wang L-D, et al. (1992) cology of G protein-linked seven transmembrane Molecular basis for the interaction of hista- 286 Molecular Medicine, Volume 1, Number 3, March 1995

mine with the . J. Biol. 11. Parker EM, Grisel DA, Iben LG, Shapiro RA. Chem. 267: 20840-20843. (1993) A single amino acid difference ac- 3. Strader CD, Sigal IS, Register RB, Candelore counts for the pharmacological distinctions MR, Rands E, Dixon RAF. (1987) Identifica- between the rat and human 5-hydroxy- tion of residues required for ligand binding tryptamine 1 B receptors. J. Neurochem. 60: to the beta-adrenergic receptor. Proc. Natl. 380-383. Acad. Sci. U.S.A. 84: 4384-4388. 12. Beinborn M, Lee Y-M, McBride EW, Quinn 4. Strader CD, Candelore MR, Hill WS, Sigal IS, SM, Kopin AS. (1993) A single amino acid of Dixon RAF. (1989) Identification of two the cholecystokinin-B/gastrin receptor de- serine residues involved in agonist activation termines for of the specificity non-peptide antago- ,B-adrenergic receptor. J. Biol. Chem. nists. Nature 362: 348-350. 264: 13572-13578. 5. Kunkel TA, Roberts JD, Zabour RA. (1987) 13. Suryanarayana S, Daunt DA, Von Zastrow Rapid and efficient site-specific mutagenesis M, Kobilka BK. (1991) A point mutation in without phenotypic selection. Methods Enzy- the seventh hydrophobic domain of the al- mol. 154: 367-382. pha 2 adrenergic receptor increases its affin- 6. Brown NA, Steofko RE, Uhler MD. (1990) ity for a family of beta receptor antagonists. Induction of alkaline phosphatase in mouse J. Biol. Chem. 266: 15488-15492. L cells by overexpression of the catalytic sub- 14. Guan X-M, Peroutka SJ, Kobilka BK. (1992) unit of cAMP-dependent protein kinase. J. Identification of a single amino acid residue Biol. Chem. 265: 13181-13189. responsible for the binding of a class of beta- 7. Chen CA, Okayama H. (1988) Calcium adrenergic receptorantagonists to 5 -hydroxy- phosphate-mediated gene transfer: A highly tryptaminelA receptors. Mol. Pharm. 41: efficient transfection system for stably trans- 695-698. forming cells with plasmid DNA. Biotech- 15. Fong TM, Yu H, Strader CD. (1992) Molec- niques 6: 632-638. ular basis for the species selectivity of the 8. Wreggett KA, Irvine RF. (1987) A rapid sep- neurokinin- 1 receptor antagonists CP-96, aration method for inositol phosphates and 345 and RP67580. J. Biol. Chem. 267: 25668- their isomers. Biochem. J. 245: 655-660. 25671. 9. DelValle J, Wang L-D, Gantz I, Schaffer M, Yamada T. (1992) Characterization of H2 his- 16. Sachais BS, Snider RM, Low JA, Krause JE. tamine receptor: Linkage to both adenylate (1993) Molecular basis for the species selec- cyclase and [Ca2+]i signaling systems. Am. J. tivity of the substance P antagonist CP-96, Physiol. 263: G967-G972. 345. J. Biol. Chem. 268: 2319-2323. 10. Oksenberg D, Marsters SA, O'Dowd BF, et 17. Gether U, Johansen TE, Snider RM, Lowe III al. (1992) A single amino-acid difference JA, Nakanishi S, Schwartz TW. (1993) Dif- confers major pharmacological variation be- ferent binding epitopes on the NK1 receptor tween human and rodent 5-HTIB receptors. for substance P and non-peptide antagonist. Nature 360: 161-163. Nature 362: 345-348.

Contributed by T. Yamada on January 12, 1995.